Abstract [en]

Isoprene (2-methylbuta-1,3-diene) is a multi-site carcinogen in rodents. To evaluate the role of the diepoxide metabolite (1,2:3,4-diepoxy-2-methylbutane) in carcinogenesis, measurements of in vivo doses of the diepoxide are needed. The in vivo dose may be inferred from levels of reaction products with hemoglobin (Hb adducts). This report presents in vitro studies of the adduct formation by the diepoxide of isoprene with valinamide and oligopeptides as model compounds of N-terminal valines in hemoglobin (Hb). In the reaction with valinamide it was shown that isoprene diepoxide forms as the main product a ring-closed adduct, which is a pyrrolidine derivative [N,N-(2,3-dihydroxy-2-methyl-1,4-butadiyl)valinamide, MPyr-Val]. The analysis was performed by gas chromatography/mass spectrometry (GC/MS) (EI and PICI) after acetylation. The ring-closed adduct was also identified by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) as the main product in the reaction between isoprene diepoxide and standard hepta- or (2H8)octapeptides, corresponding to the N-terminal peptides of the α-chains in mouse and rat Hb. These peptides, alkylated with isoprene diepoxide, to be used as internal standards and calibration standards for quantification of MPyr-adduct levels in vitro and in vivo, were analyzed with respect to the degree of MPyr-alkylation by two independent methods, amino acid analysis and HPLC-UV; similar results were obtained using these methods. A method for measurement of Hb adducts as modified peptides, used earlier to measure a similar adduct to N-terminal valines in Hb from the diepoxide of 1,3-butadiene, has in the present work been tested for application to isoprene diepoxide. The method is based on tryptic degradation of globin and LC/ESI-MS analysis of N-terminal Pyr-heptapeptides of the Hb α-chain enriched by HPLC. MPyr-adduct levels in isoprene diepoxide alkylated hemolysate from mouse erythrocytes incubated with different concentrations of isoprene diepoxide (2 and 10 mM) for 1 h were quantified. The adduct level was about 50 nmol/g α-chain Hb per mM × h. From the adduct levels the rate constant of isoprene diepoxide for reaction with N-terminal valine was calculated to be about 1.6 times faster than for diepoxybutane

Fred, Charlotta

Abstract [en]

1,3-Butadiene is a general air pollutant associated with combustion of organic matter and is also an extensively used monomer in polymer production. The cancer risk estimation of 1,3-butadiene is encumbered with large uncertainties. Extrapolation from tumour frequencies in long-term animal tests has led to a relatively high figure for the risk associated with 1,3-butadiene exposure. This is mainly based on observations of very high tumour incidences in butadiene-exposed mice, which in this respect are about 100 times more sensitive than rats. It has been hypothesized that a high cancer risk from 1,3-butadiene could be associated with its metabolism to the bifunctional 1,2:3,4-diepoxybutane (DEB) which, in comparison with monofunctional epoxides, 1,2-epoxy-3-butene (EB) and 1,2-epoxy-3,4-butanediol (EBdiol), is a highly effective mutagen, i.e. cancer initiator. Measurement of in vivo doses of DEB is therefore essential for the risk assessment of 1,3-butadiene. Reaction products with hemoglobin offer a possibility of measuring reactive metabolites in vivo. Hemoglobin adducts from EBdiol have in this study been measured with available methods, which are, however, not applicable to the bifunctional DEB, and method development was therefore needed.

This work presents a procedure for measurement of a specific, ring-closed adduct, Pyr-Val, formed from the reaction of DEB with N-terminal valines in hemoglobin. It is based on LC-ESI-MS/MS analysis of the Pyr-modified N-terminal peptides enriched after trypsin digestion of globin. Mouse and rat could be compared regarding the metabolism of EB, DEB and EBdiol. From the data it was concluded that, in 1,3-butadiene exposure, about 60 times higher levels of DEB are formed in mice compared to rats. Estimates of in vivo doses in published cancer tests showed that carcinogenesis in mice is mainly due to DEB, whereas in rat, and possibly man, the monofunctional EBdiol is the predominant causative factor. Preliminarily, the cancer risk assessed from these data is compatible with the epidemiology-based risk estimate of US EPA.

Due to the structural similarity with 1,3-butadiene, certain parallel studies of isoprene (2-methyl-1,3-butadiene) metabolism were initiated. Isoprene is the major endogenously produced hydrocarbon in humans and mammals and shows a similar difference in sensitivity between species for tumour development as 1,3-butadiene. In mice treated with the isoprene monoepoxide, 1,2-epoxy-2-methyl-3-butene (IMO), an in vivo formation of the corresponding diepoxide, 1,2:3,4-diepoxy-2-methyl-butane (IDO), was demonstrated. The in vivo dose of IDO formed from IMO was about half of that of DEB formed from EB. In the analysis of bone marrow erythrocytes an increased frequency of micronuclei, induced by treatment with EB or IMO, showed correlation with the in vivo doses of the respective diepoxides.

With the ambition to reduce animal experiments a general procedure has been developed for trapping reactive metabolites in in vitro test systems, with the specific aim to study differences between species in metabolism of 1,3-butadiene. Vitamin B12 in its reduced form [Cbl(I)] has been used for instant trapping of 1,3-butadiene metabolites formed in S9-mixture. LC-ESI-MS/MS is then used for quantification of the formed alkyl-Cbls. The method has been applied to the epoxide metabolites of 1,3-butadiene, which all form specific alkyl-Cbls in the reaction with Cbl(I)